"ISA-Lation" of Single-Stranded Positive-Sense RNA Viruses from Non-Infectious Clinical/Animal Samples.

Lisa F.P. Ng,Fabien Aubry,Ernest A. Gould,Antoine Nougairède,Xavier de Lamballerie,Géraldine Piorkowski,Lauriane de Fabritus

doi:10.1371/journal.pone.0138703

Abstract

Isolation of viral pathogens from clinical and/or animal samples has traditionally relied on either cell cultures or laboratory animal model systems. However, virus viability is notoriously susceptible to adverse conditions that may include inappropriate procedures for sample collection, storage temperature, support media and transportation. Using our recently described ISA method, we have developed a novel procedure to isolate infectious single-stranded positive-sense RNA viruses from clinical or animal samples. This approach, that we have now called "ISA-lation", exploits the capacity of viral cDNA subgenomic fragments to re-assemble and produce infectious viral RNA in susceptible cells. Here, it was successfully used to rescue enterovirus, Chikungunya and Tick-borne encephalitis viruses from a variety of inactivated animal and human samples. ISA-lation represents an effective option to rescue infectious virus from clinical and/or animal samples that may have deteriorated during the collection and storage period, but also potentially overcomes logistic and administrative difficulties generated when complying with current health and safety and biosecurity guidelines associated with shipment of infectious viral material.

Highlights

Isolation of viruses using cell culture or laboratory animals has, for many years, been the “gold standard” for human and veterinarian clinical virology laboratories [1,2] and for environmental virology studies [3]
Next-Generation Sequencing (NGS) complete genomic sequencing confirmed the integrity of the genome structure and the genetic similarity with the tick-borne encephalitis virus (TBEV) Oshima 5–10 strain used for infecting mice (Tables D and E in S1 File)
We have developed a technically simple but scientifically sophisticated procedure designated “infectious subgenomic amplicons (ISA)-lation” which facilitates the rescue of infectious single-stranded positive-sense RNA viruses from clinical samples in which, for a variety of reasons, virus infectivity has been lost but genomic RNA has retained sufficient fidelity to enable authentic RT-PCR amplification

Summary

Introduction

Isolation of viruses using cell culture or laboratory animals has, for many years, been the “gold standard” for human and veterinarian clinical virology laboratories [1,2] and for environmental virology studies [3]. The isolation of viral pathogens requires infectious virus to be present in the sample. Viability can readily be compromised due to intrinsic fragility of the virus, inappropriate sample collection, unsuitable transportation and/or storage conditions, or PLOS ONE | DOI:10.1371/journal.pone.0138703. "ISA-Lation" of RNA Viruses from Clinical/Animal Samples recherche.fr/ (grant ANR-14-CE14-0001 RNA VacciCode). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion